Biology subjects

Huang, C.

Publications and source records attributed to Huang, C..

At least 163 records · Page 9Linked to original sources

bioRxiv2022

Thalamo-Prefrontal Connectivity Correlates with Early Command-Following After Severe Traumatic Brain injury

Recovery of consciousness after traumatic brain injury (TBI) is heterogeneous and difficult to predict. Structures such as the thalamus and prefrontal cortex are thought to be important in facilitating consciousness. We sought to investigate whether the integrity of thalamo-prefrontal circuits, assessed via diffusion tensor imaging (DTI), was associated with the return of goal-directed behavior after severe TBI. We classified a cohort of severe TBI patients (N = 25, 20 males) into Early and Late/Never outcome groups based on their ability to follow commands within 30 days post-injury. We assessed connectivity between whole thalamus, and mediodorsal thalamus (MD), to prefrontal cortex (PFC) subregions including dorsolateral PFC (dlPFC), medial PFC (mPFC), anterior cingulate (ACC), and orbitofrontal (OFC) cortices. We found that the integrity of thalamic projections to PFC subregions (L OFC, L and R ACC, and R mPFC) was significantly associated with Early command-following. This association persisted when the analysis was restricted to prefrontal-mediodorsal (MD) thalamus connectivity. In contrast, dlPFC connectivity to thalamus was not significantly associated with command-following. Using the integrity of thalamo-prefrontal connections, we created a linear regression model that demonstrated 72% accuracy in predicting command-following after a leave-one-out analysis. Together, these data support a role for thalamo-prefrontal connectivity in the return of goal-directed behavior following TBI.

neuroscience↗

bioRxiv2021

Comprehensive transcriptional profiling and mouse phenotyping reveals dispensable role for adipose tissue selective long noncoding RNA Gm15551

1Cold and nutrient activated brown adipose tissue (BAT) is capable of increasing systemic energy expenditure via uncoupled respiration and secretion of endocrine factors thereby protecting mice against diet-induced obesity and improving insulin response and glucose tolerance in men. Long non-coding RNAs (lncRNAs) have recently been identified as fine tuning regulators of cellular function. While certain lncRNAs have been functionally characterised in adipose tissue, their overall contribution in the activation of BAT remains elusive. We identified lncRNAs correlating to inter-scapular brown adipose tissue (iBAT) function in high fat diet (HFD) and cold stressed mice. We focused on Gm15551 which has an adipose tissue specific expression profile, is highly upregulated during adipogenesis and downregulated by {beta}-adrenergic activation in mature adipocytes. Albeit we performed comprehensive transcriptional and adipocyte physiology profiling in vitro and in vivo, we could not detect an effect of gain or loss of function of Gm15551.

molecular biology↗

bioRxiv2021

METTL3 facilitates hepatic fibrosis progression via m6A-YTHDF2 dependent silencing of GPR161

Hepatic fibrosis (HF) is a very common condition seen in millions of patients with various liver diseases. N6-methyladenosine (m6A) plays critical roles in various biological and pathological processes. However, the role of m6A and its main methyltransferase METTL3 in HF remains obscure. Here, we reported that METTL3 expression was elevated in HSCs from CCl4 induced fibrotic liver. METTL3 knockdown in HSCs mediated by recombinant adeno-associated-virus serotype 9 packed short hairpin RNA against METTL3 alleviated liver injury and fibrosis compared to empty carrier group. Mechanistically, the decreased liver fibrosis in CCl4-treated HSC-specific METTL3 knockdown mice was due to the increased GPR161 that is a suppressor of Hedgehog pathway, a well-known pathway to activate in liver injury and regeneration. As expect, GPR161 transferred into HSCs alleviated liver fibrosis and HSC activation. Forced GPR161 expression inhibited Gli3 activated form nuclear accumulation and subsequently suppressed fibrosis-associate gene expression. Conclusion, HSC-specific deletion of METTL3 inhibits liver fibrosis via elevated GPR161 expression, which subsequently suppressed Hedgehog pathway activation and fibrosis-associated genes expression, providing novel therapeutic targets for HF therapy. Synopsis O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/472749v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@1245f3aorg.highwire.dtl.DTLVardef@e7effdorg.highwire.dtl.DTLVardef@13ce319org.highwire.dtl.DTLVardef@61be8b_HPS_FORMAT_FIGEXP M_FIG C_FIG The role of RNA m6A methyltransferase METTL3 in the progression of hepatic fibrosis is unclear. Here, we reveal that suppresses m6A modification by depletion of METTL3 in Hepatic Stellate Cells (HSCs), stabilizes the GPR161 transcripts, that involved in suppressing Hedgehog signaling pathway, through YTHDF2, thereby alleviating the fibrosis feature of CCl4-induced liver injury. O_LIDisruption of m6A methyltransferases METTL3 in HSCs alleviates liver fibrosis feature. C_LIO_LIMETTL3 depletion in liver HSCs suppresses its activation by suppressing Hedgehog signaling pathway through stabilizing GPR161 transcripts in an m6A-YTHDF2 dependent manner. C_LIO_LIGPR161 activates PKA that mediates phosphorylation and degradation of full-length Gli3 (Gli3-FL) protein, then the short Gli3 (Gli3-R) translocated into the nucleus and suppresses the fibrosis associated gene transcription. C_LIO_LIThe elevated METTL3 expression is attribute to its promoter H3K27ac modification. C_LI

molecular biology↗

bioRxiv2021

Models for Evaluation of Biological Age Based on Multi-omics

Aging is a complex process of systemic degeneration at multiple cellular and tissue levels that has a complex mechanism. Individuals age at different rates, and there is a high degree of aging heterogeneity. Therefore, it is inaccurate to judge an individuals degree of aging by their chronological age. We performed transcriptome-focused multi-omics analyses (including transcriptomics, DNA methylation, proteomics, cytokine and telomere analysis, and single-cell transcriptome sequencing) on 139 healthy individuals aged 23 to 88 years. We systematically analyzed linear and non-linear changes in gene expression throughout aging. Genes with similar expression trajectories were enriched in similar biological pathways, including the PI3K-AKT signaling pathway and the inflammatory response. Through DNA methylation detection, we found that the expression of the top genes correlated with age was affected by methylation in the gene promoter region. These genes had no significant correlation with the expression of downstream proteins, but they were enriched in PI3K-AKT-associated proteins. Single-cell transcriptome sequencing showed that the expression of these genes did not significantly change in different cell subtypes, which proves that the gene expression changes were caused by internal age-related cellular changes rather than cell composition changes. We designed a transcriptome age clock and a methylation age clock using a set of 787 genes. Our models can accurately predict age with a mean absolute error (MAE) of 5.203 and 3.28, respectively, which is better than previously established aging models. The accuracy of our model was further verified by the detection of telomeres, which can identify accelerated aging of individuals.

genomics↗

bioRxiv2021

Machupo virus with mutations in the transmembrane domain and glycosylation sites is attenuated and immunogenic in animal models of Bolivian Hemorrhagic Fever

Several highly pathogenic mammarenaviruses cause severe hemorrhagic and neurologic disease in humans, for which vaccines and antivirals are limited or unavailable. New World (NW) mammarenavirus Machupo virus (MACV) infection causes Bolivian hemorrhagic fever in humans. We previously reported that the disruption of specific N-linked glycan sites on the glycoprotein (GPC) partially attenuate MACV in an IFN-{beta}/{gamma} receptor knockout mouse model. However, some capability to induce neurological pathology still remained. Highly pathogenic Junin virus (JUNV) is another NW arenavirus closely related to MACV. A F427I substitution in the GPC transmembrane domain (TMD) rendered JUNV attenuated in a lethal mouse model after intracranial inoculation. In this study, we rationally designed and rescued a MACV containing mutations at two glycosylation sites and the corresponding F438I substitution in GPC TMD. The MACV mutant is fully attenuated in IFN-{beta}/{gamma} receptor knockout mice and outbred guinea pigs. Furthermore, inoculation with this mutant MACV fully protected guinea pigs from wild-type MACV lethal challenge. Lastly, we found the GPC TMD F438I substitution greatly impaired MACV growth in neuronal cell lines of mouse and human origins. Our results highlight the critical roles of the glycans and the TMD on the GPC in arenavirus virulence, which informs the rational design of potential vaccine candidates for highly pathogenic arenaviruses. ImportanceFor arenaviruses, the only vaccine available is the live-attenuated Candid#1 vaccine, a JUNV vaccine approved in Argentina. We and others have found that the glycans on GPC and the F427 residue in the GPC TMD are important for virulence of JUNV. Nevertheless, mutating either of them is not sufficient for full and stable attenuation of JUNV. Using reverse genetics, we disrupted specific glycosylation sites on MACV GPC, and also introduced the corresponding F438I substitution in the GPC TMD. This MACV mutant is fully attenuated in two animal models and protects animals from lethal infection. Thus, our studies highlight the feasibility of rational attenuation of highly pathogenic arenaviruses for vaccine development. Another important finding from this study is that the F438I substitution in GPC TMD could substantially affect MACV replication in neurons. Future studies are warranted to elucidate the underlying mechanism and the implication of this mutation in arenavirus neural tropism.

microbiology↗

bioRxiv2021

A Complete Pedigree-Based Graph Workflow for Rare Candidate Variant Analysis

Methods that use a linear genome reference for genome sequencing data analysis are reference biased. In the field of clinical genetics for rare diseases, a resulting reduction in genotyping accuracy in some regions has likely prevented the resolution of some cases. Pangenome graphs embed population variation into a reference structure. While pangenome graphs have helped to reduce reference mapping bias, further performance improvements are possible. We introduce VG-Pedigree, a pedigree-aware workflow based on the pangenome-mapping tool of Giraffe (Siren et al. 2021) and the variant-calling tool DeepTrio (Kolesnikov et al. 2021) using a specially-trained model for Giraffe-based alignments. We demonstrate mapping and variant calling improvements in both single-nucleotide variants (SNVs) and insertion and deletion (INDEL) variants over those produced by alignments created using BWA-MEM to a linear-reference and Giraffe mapping to a pangenome graph containing data from the 1000 Genomes Project. We have also adapted and upgraded the deleterious-variant (DV) detecting methods and programs of Gu et al. into a streamlined workflow (Gu et al. 2019). We used these workflows in combination to detect small lists of candidate DVs among 15 family quartets and quintets of the Undiagnosed Diseases Program (UDP). All candidate DVs that were previously diagnosed using the mendelian models covered by the previously published Gu et al. methods were recapitulated by these workflows. The results of these experiments indicate a slightly greater absolute count of DVs are detected in the proband population than in their matched unaffected siblings.

bioinformatics↗

bioRxiv2021

Three-dimensional Shear Wave Elastography Using a 2D Row Column Addressing (RCA) Array

ObjectiveTo develop a 3D shear wave elastography (SWE) technique using a 2D row column addressing (RCA) array, with either external vibration or acoustic radiation force (ARF) as the shear wave source. Impact StatementThe proposed method paves the way for clinical translation of 3D-SWE based on the 2D RCA, providing a low-cost and high volume-rate solution that is compatible with existing clinical systems. IntroductionSWE is an established ultrasound imaging modality that provides a direct and quantitative assessment of tissue stiffness, which is significant for a wide range of clinical applications including cancer and liver fibrosis. SWE requires high frame-rate imaging for robust shear wave tracking. Due to the technical challenges associated with high volume-rate imaging in 3D, current SWE techniques are typically confined to 2D. Advancing SWE from 2D to 3D is significant because of the heterogeneous nature of tissue, which demands 3D imaging for accurate and comprehensive evaluation. MethodsA 3D SWE method using a 2D RCA array was developed with a volume-rate up to 2000 Hz. The performance of the proposed method was systematically evaluated on tissue-mimicking elasticity phantoms. Results3D shear wave motion induced by either external vibration or ARF was successfully detected with the proposed method. Robust 3D shear wave speed maps were reconstructed for both homogeneous and heterogeneous phantoms with inclusions. ConclusionThe high volume-rate 3D imaging provided by the 2D RCA array provides a robust and practical solution for 3D SWE with a clear pathway for future clinical translation.

bioengineering↗

bioRxiv2021

Determining the architecture of nuclear ring of Xenopus laevis nuclear pore complex using integrated approaches

The nuclear pore complexes (NPCs) are large protein assemblies as a physical gate to regulate nucleocytoplasmic transport. Here, using integrated approaches including cryo-electron microscopy, hybrid homology modeling and cell experiment, we determined the architecture of the nuclear ring (NR) from Xenopus laevis oocytes NPC at subnanometer resolution. In addition to the improvement of the Y complex model, eight copies of Nup205 and ELYS were assigned in NR. Nup205 connects the inner and outer Y complexes and contributes to the assembly and stability of the NR. By interacting with both the inner Nup160 and the nuclear envelope (NE), the N-terminal {beta}-propeller and -solenoid domains of ELYS were found to be essential for accurate assembly of the NPC on the NE.

molecular biology↗

bioRxiv2021

The cytosolic role of EZH2-IMPDH2 complex in melanoma progression and metastasis via GTP regulation

The enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/467024v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@153dd35org.highwire.dtl.DTLVardef@dfeb43org.highwire.dtl.DTLVardef@ce8148org.highwire.dtl.DTLVardef@189d8da_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG HighlightsO_LIEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma. C_LIO_LIThe N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner. C_LIO_LIEZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2s tetramerization-mediated activity independently of EZH2 methyltransferase. C_LIO_LIEZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase. C_LIO_LISappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells. C_LI

cancer biology↗

bioRxiv2021

Multi-omics analysis for potential inflammation-related genes involved in tumor immune evasion via extended application of epigenetic data

BackgroundAccumulating evidence suggests that inflammation-related genes may play key roles in tumor immune evasion. Programmed cell death ligand 1 (PD-L1) is an important immune checkpoint involved in mediating antitumor immunity. We performed multi-omics analysis to explore key inflammation-related genes affecting the transcriptional regulation of PD-L1 expression. MethodsThe open chromatin region of the PD-L1 promoter was mapped using the assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles. Correlation analysis of epigenetic data (ATAC-seq) and transcriptome data (RNA-seq) were performed to identify inflammation-related transcription factors whose expression levels were correlated with the chromatin accessibility of the PD-L1 promoter. Chromatin immunoprecipitation sequencing (ChIP-seq) profiles were used to confirm the physical binding of the TF STAT2 and the predicted binding regions. We also confirmed the results of the bioinformatics analysis with cell experiments. ResultsWe identified chr9:5449463-5449962 and chr9:5450250-5450749 as reproducible open chromatin regions in the PD-L1 promoter. Moreover, we observed a correlation between STAT2 expression and the accessibility of the aforementioned regions. Furthermore, we confirmed its physical binding through ChIP-seq profiles and demonstrated the regulation of PD-L1 by STAT2 overexpression in vitro. Multiple databases were also used for the validation of the results. ConclusionOur study identified STAT2 as a direct upstream TF regulating PD-L1 expression. The interaction of STAT2 and PD-L1 might be associated with tumor immune evasion in cancers, suggesting the potential value for tumor treatment.

immunology↗

bioRxiv2021

Suppression of a dsRNA-induced plant immunity pathway by viral movement protein

The virome of plants is dominated by RNA viruses 1 and several of these cause devastating diseases in cultivated plants leading to global crop losses 2. To infect plants, RNA viruses engage in complex interactions with compatible plant hosts. In cells at the spreading infection front, RNA viruses replicate their genome through double-stranded RNA (dsRNA) intermediates and interact with cellular transport processes to achieve cell-to-cell movement of replicated genome copies through cell wall channels called plasmodesmata (PD) 3. In order to propagate, viruses also must overcome host defense responses. In addition to triggering the antiviral RNA silencing response, RNA virus infection also elicits pattern-triggered immunity (PTI) 4 whereby dsRNA, a hallmark of virus replication, acts as an important elicitor 5. This innate antiviral immune response is also triggered when dsRNA is applied externally and does not require sequence homology to the virus 5. However, the mechanism by which PTI restricts virus infection is not known. Here, we show that dsRNA inhibits the progression of virus infection by triggering callose deposition at plasmodesmata and the inhibition of transport through these cell-to-cell communication channels. The dsRNA-induced signaling pathway leading to callose deposition is independent of ROS production and thus distinguished from pathways triggered by bacterial and fungal elicitors. The dsRNA-induced host response at plasmodesmata is suppressed by the Tobacco mosaic virus movement protein (MP). Thus, the virus uses MP to inhibit innate dsRNA-induced immunity at plasmodesmata, which could be a general strategy of phytoviruses to overcome plant defenses and spread infection.

plant biology↗

bioRxiv2021

Dual polarity voltage imaging reveals subthreshold dynamics and concurrent spiking patterns of multiple neuron-types

Genetically encoded fluorescent voltage indicators are ideally suited to reveal the millisecond-scale interactions among and between distinct, targeted cell populations. However, current indicator families lack the requisite sensitivity for in vivo multipopulation imaging. We describe high-performance green and red sensors, Ace-mNeon2 and VARNAM2, and their reverse response-polarity variants, pAce and pAceR. Our indicators enable 0.4-1 kHz voltage recordings from >50 neurons per field-of-view in awake mice and [~]30-min continuous imaging in flies. Using dual-polarity multiplexed imaging, we uncovered behavioral state-dependent interactions between distinct neocortical subclasses, as well as contributions to hippocampal field potentials from non-overlapping projection neuronal ensembles. By combining three mutually compatible indicators, we demonstrate concurrent triple-population voltage imaging. Our approach will empower investigations of the dynamic interplay between neuronal subclasses at single-spike resolution. One Sentence SummaryA new suite of voltage sensors enables simultaneous cellular-resolution activity imaging from multiple, targeted neuron-types in awake animals.

neuroscience↗

bioRxiv2021

Elevated transcriptional pausing of RNA polymerase II underlies acquired resistance to radiotherapy

As the mainstay modality for many malignancies, particularly inoperable solid tumors such as nasopharyngeal carcinoma (NPC), ionizing radiation (IR) induces a variety of lesions in genomic DNA, evoking a multipronged DNA damage response to interrupt many cellular processes including transcription. The turbulence in transcription, depending on the nature of DNA lesions, encompasses local blockage of RNA polymerase II (RNAPII) near the damage sites, as well as a less understood genome-wide alteration. How the transcriptional change influences the effectiveness of radiotherapy remains unclear. Using a panel of NPC and lung cancer cell lines, we observe increased phosphorylation at serine 5 (pS5) of the RNAPII after IR, indicating an accumulation of paused RNAPII. Remarkably, a similar increase of pS5 is seen in IR-resistant cells. ChIP-seq analysis of RNAPII distribution confirms this increased pausing both in IR-treated and IR-resistant NPC cells, notably on genes involved in radiation response and cell cycle. Accordingly, many of these genes show downregulated transcripts abundance in IR-resistant cells, and individual knockdown some of them such as TP53 and NEK7 endows NPC cells with varying degrees of IR-resistance. Decreasing pS5 of RNAPII and hence tuning down transcriptional pausing by inhibiting CDK7 reverses IR-resistance both in cell culture and xenograft models. Our results therefore uncover an unexpected link between elevated transcriptional pausing and IR-resistance. Given the recurrent NPC tissues display a steady increase in pS5 compared to the paired primary tissues, we suggest that CDK7 inhibitors can be used in combination with radiotherapy to increase sensitivity and thwart resistance.

cancer biology↗

bioRxiv2021

Ubr1-induced selective endo-phagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The defence mechanisms against endo-lysosomal homeostasis stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) ubiquitin ligase that counteracts proteostasis stress by enhancing cargo selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations increased intracellular Ca2+ and caused endosomal compartment stress by fusion and enlargement. Endosomal protein QC pathway using ubiquitin QC ligases CHIP and Ubr1 with ESCRT-machinery was able to target only a fraction of MLC1-mutants for lysosomal degradation. As a consequence of the endosomal stress, we found an alternative QC route dependent on Ubr1, SQSTM1/p62 and arginylation to bypass MLC1-mutants to endosomal autophagy (endo-phagy). Significantly, this unfolded a general biological endo-lysosomal QC pathway for arginylated Ubr1-SQSTM1/p62 autophagy targets during Ca2+-assault. Conversely, the loss of Ubr1 with the absence of arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1-dependent endo-phagy/autophagy in constitutive and provoked endo-lysosomal proteostasis stress, and link Ubr1 to Ca2+-homeostasis and proteins implicated in various diseases including cancers and brain disorders.

biochemistry↗

bioRxiv2021

Localization free super-resolution microbubble velocimetry using a long short-term memory neural network

Ultrasound localization microscopy is a super-resolution imaging technique that exploits the unique characteristics of contrast microbubbles to side-step the fundamental trade-off between imaging resolution and penetration depth. However, the conventional reconstruction technique is confined to low microbubble concentrations to avoid localization and tracking errors. Several research groups have introduced sparsity- and deep learning-based approaches to overcome this constraint to extract useful vascular structural information from overlapping microbubble signals, but these solutions have not been demonstrated to produce blood flow velocity maps of the microcirculation. Here, we introduce Deep-SMV, a localization free super-resolution microbubble velocimetry technique, based on a long short-term memory neural network, that provides high imaging speed and robustness to high microbubble concentrations, and directly outputs blood velocity measurements at a super-resolution. Deep-SMV is trained efficiently using microbubble flow simulation on real in vivo vascular data and demonstrates real-time velocity map reconstruction suitable for functional vascular imaging and pulsatility mapping at super-resolution. The technique is successfully applied to a wide variety of imaging scenarios, include flow channel phantoms, chicken embryo chorioallantoic membranes, and mouse kidney, tumor, and brain imaging.

bioengineering↗

bioRxiv2021

Topological insights into the neural basis of flexible behavior

It is widely accepted that there is an inextricable link between neural computations, biological mechanisms, and behavior, but it is challenging to simultaneously relate all three. Here, we show that topological data analysis (TDA) provides an important bridge between these approaches to studying how brains mediate behavior. We demonstrate that cognitive processes change the topological description of the shared activity of populations of visual neurons. These topological changes constrain and distinguish between competing mechanistic models, are connected to subjects performance on a visual change detection task, and, via a link with network control theory, reveal a tradeoff between improving sensitivity to subtle visual stimulus changes and increasing the chance that the subject will stray off task. These connections provide a blueprint for using TDA to uncover the biological and computational mechanisms by which cognition affects behavior in health and disease. Significance StatementAs the fields of systems, computational, and cognitive neuroscience strive to establish links between computations, biology, and behavior, there is an increasing need for an analysis framework to bridge levels of analysis. We demonstrate that topological data analysis (TDA) of the shared activity of populations of neurons provides that link. TDA allows us to distinguish between competing mechanistic models and to answer longstanding questions in cognitive neuroscience, such as why there is a tradeoff between visual sensitivity and staying on task. These results and analysis framework have applications to many systems within neuroscience and beyond.

neuroscience↗

bioRxiv2021

Effect of ORF7 of SARS-CoV-2 on the chemotaxis of monocytes and neutrophils in vitro

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most significant public health threats in worldwide. Patients with severe COVID-19 usually have pneumonia concomitant with local inflammation and sometimes a cytokine storm. Specific components of the SARS-CoV-2 virus trigger lung inflammation, and recruitment of immune cells to the lungs exacerbates this process, although much remains unknown about the pathogenesis of COVID-19. Our study of lung type II pneumocyte cells (A549) demonstrated that ORF7, an open reading frame (ORF) in the genome of SARS-CoV-2, induced the production of CCL2, a chemokine that promotes the chemotaxis of monocytes, and decreased the expression of IL-8, a chemokine that recruits neutrophils. A549 cells also had an increased level of IL-6. The results of our chemotaxis transwell assay suggested that ORF7 augmented monocyte infiltration and reduced the number of neutrophils. We conclude that the ORF7 of SARS-CoV-2 may have specific effects on the immunological changes in tissues after infection. These results suggest that the functions of other ORFs of SARS-CoV-2 should also be comprehensively examined.

cell biology↗

bioRxiv2021

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an -methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459913v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f4764org.highwire.dtl.DTLVardef@12818daorg.highwire.dtl.DTLVardef@177bdf0org.highwire.dtl.DTLVardef@57046c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗